PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.
Keywords: cell model; hPHT2; substrates; transport function.
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